The N-terminal TOG domain of Arabidopsis MOR1 modulates affinity for microtubule polymers
The N-terminal TOG domain of Arabidopsis MOR1 modulates affinity for microtubule polymers
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DOI：
10.1242/jcs.107045
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发表时间：
2012-10-15
影响因子：
4
通讯作者：
Wasteneys, Geoffrey O.
中科院分区：
文献类型：
--
作者：
Lechner, Bettina;Rashbrooke, Madeleine C.;Wasteneys, Geoffrey O.
高度保守的XMAP215/Dis1家族的微管相关蛋白促进微管的生长和收缩，并随动态微管末端移动。植物同源物MOR1被预测形成一个具有5个N-末端TOG结构域的长线性分子。在第一个结构域(TOG1)内，mor1-1亮氨酸到苯丙氨酸(L174F)的取代导致微管阵列随温度变化而解体，并降低微管的生长和收缩速度。通过在植物中表达MOR1的两个N-末端TOG结构域(TOG12)，以便在活细胞中分析，以及在体外微管结合和聚合实验中的细菌中的表达，我们确定MOR1的N-末端结构域对微管聚合物结合是至关重要的。当TOG12在拟南芥中稳定表达或在韭菜表皮细胞中瞬时过表达时，在N端标记TOG12会干扰其结合微管的能力，并阻碍体外聚合酶的活性。相反，标记在C端的TOG12在体内与微管相互作用，挽救了对温度敏感的Mor1-1表型，并在体外促进了微管聚合。含有L174F Mor1-1点突变的TOG12构建体在大蒜表皮瞬时过表达时会导致微管破裂，并在体外增加TOG12与微管的亲和力。这表明，Mor1-1突变蛋白通过过于强烈地结合微管晶格而使微管变得不那么动态，从而无法支持快速加端跟踪。根据我们的结果，我们得出结论，N端TOG结构域中平衡的微管亲和力对MOR1的聚合酶活性至关重要。
Microtubule-associated proteins of the highly conserved XMAP215/Dis1 family promote both microtubule growth and shrinkage, and move with the dynamic microtubule ends. The plant homologue, MOR1, is predicted to form a long linear molecule with five N-terminal TOG domains. Within the first (TOG1) domain, the mor1-1 leucine to phenylalanine (L174F) substitution causes temperature-dependent disorganization of microtubule arrays and reduces microtubule growth and shrinkage rates. By expressing the two N-terminal TOG domains (TOG12) of MOR1, both in planta for analysis in living cells and in bacteria for in vitro microtubule-binding and polymerization assays, we determined that the N-terminal domain of MOR1 is crucial for microtubule polymer binding. Tagging TOG12 at the N-terminus interfered with its ability to bind microtubules when stably expressed in Arabidopsis or when transiently overexpressed in leek epidermal cells, and impeded polymerase activity in vitro. In contrast, TOG12 tagged at the C-terminus interacted with microtubules in vivo, rescued the temperature-sensitive mor1-1 phenotype, and promoted microtubule polymerization in vitro. TOG12 constructs containing the L174F mor1-1 point mutation caused microtubule disruption when transiently overexpressed in leek epidermis and increased the affinity of TOG12 for microtubules in vitro. This suggests that the mor1-1 mutant protein makes microtubules less dynamic by binding the microtubule lattice too strongly to support rapid plus-end tracking. We conclude from our results that a balanced microtubule affinity in the N-terminal TOG domain is crucial for the polymerase activity of MOR1.